Maintenance of Leukemia-Initiating Cells Is Regulated by the CDK Inhibitor Inca1

Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of Inca1 led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, Inca1-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in Inca1-deficient cells in vivo. As a consequence, leukemia induction and leukemia maintenance were severely impaired in Inca1−/− bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of Inca1−/− in MLL—AF9- and c-myc/BCL2-positive leukemia mouse models. These findings indicate distinct functional properties of Inca1 in normal hematopoietic cells compared to leukemia initiating cells. Such functional differences might be used to design specific therapy approaches in leukemia

Maintenance of Leukemia-Initiating Cells Is Regulated by the CDK Inhibitor Inca1

<div><p>Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of <i>Inca1</i> led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, <i>Inca1</i>-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in <i>Inca1</i>-deficient cells <i>in vivo</i>. As a consequence, leukemia induction and leukemia maintenance were severely impaired in <i>Inca1^−/−</i> bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of <i>Inca1^−/−</i> in MLL—AF9- and c-myc/BCL2-positive leukemia mouse models. These findings indicate distinct functional properties of Inca1 in normal hematopoietic cells compared to leukemia initiating cells. Such functional differences might be used to design specific therapy approaches in leukemia.</p></div

Eph-ephrin signaling couples endothelial cell sorting and arterial specification

Abstract Cell segregation allows the compartmentalization of cells with similar fates during morphogenesis, which can be enhanced by cell fate plasticity in response to local molecular and biomechanical cues. Endothelial tip cells in the growing retina, which lead vessel sprouts, give rise to arterial endothelial cells and thereby mediate arterial growth. Here, we have combined cell type-specific and inducible mouse genetics, flow experiments in vitro, single-cell RNA sequencing and biochemistry to show that the balance between ephrin-B2 and its receptor EphB4 is critical for arterial specification, cell sorting and arteriovenous patterning. At the molecular level, elevated ephrin-B2 function after loss of EphB4 enhances signaling responses by the Notch pathway, VEGF and the transcription factor Dach1, which is influenced by endothelial shear stress. Our findings reveal how Eph-ephrin interactions integrate cell segregation and arteriovenous specification in the vasculature, which has potential relevance for human vascular malformations caused by EPHB4 mutations

Cytotoxic stress exhausts the stem cell pool in <i>Inca1</i>-deficient bone marrow.

<p><b>A.</b> The Kaplan-Meier plot illustrates the survival of <i>Inca1^+/+</i> (n = 7) and <i>Inca1^−/−</i> (n = 13) mice after weekly administration of the myeloablative agent 5-fluorouracil (5-FU). 5-FU treatment targeted predominantly proliferating bone marrow cells and led to a significantly shortened lifespan of <i>Inca1^−/−</i> mice compared to their wild type littermates (p = 0.02, log-rank). <b>B.</b> Cell cycle analysis of total bone marrow from mice treated twice with 5-FU using BrdU/PI staining and subsequent FACS analysis. <i>Inca1^−/−</i> bone marrow cells of mice that had been exposed twice to 5-FU showed more cells in S-phase than 5-FU treated wild type mice. Shown here is a representative example of three independent experiments. <b>C.</b> Histological bone sections from 5-FU treated mice showed significant depletion of hematopoietic cells in the bone marrow of <i>Inca1^−/−</i> sternum (right-hand side) with replacement by fat cells compared to wild type control mice. In contrast, hematopoietic cell numbers were only mildly decreased in wild type mice (left panel). Lower panels show higher magnifications of the areas marked in the upper panels. <b>D.</b> The total number of nucleated bone marrow cells was significantly decreased in <i>Inca1^−/−</i> mice after two cycles of 5-FU treatment (mean ± SD, p = 0.002, t-test). All mice were 16 weeks old at the time of analysis. <b>E.</b> Bone marrow cellularity in non-challenged mice did not differ between <i>Inca1^−/−</i> and <i>Inca1^+/+</i> genotypes (mice aged 70 to 425 days; mean ± SD, p = 0.83, t-test).</p

Self-renewal of AML1-ETO9a-transduced bone marrow cells is impaired in absence of <i>Inca1</i>.

<p><b>A.</b> Schematic overview about the performed transduction and transplantation experiments. Bone marrow isolated from <i>Inca1^+/+</i> or <i>Inca1^−/−</i> mice was retrovirally transduced with AML1-ETO9a-IRES-GFP, MLL-AF9-IRES-GFP, or c-myc-IRES-BCL2-IRES-mCherry. Equal numbers of positive cells were transplanted into lethally irradiated recipients, which were then subjected to different analyses. <b>B.</b> Engraftment of AML1-ETO9a-transduced <i>Inca1^+/+</i> and <i>Inca1^−/−</i> bone marrow cells was determined by FACS analysis of GFP-positive cells in the blood of transplanted mice from 4 to 40 weeks. Engraftment was initially higher in recipients transplanted with <i>Inca1^−/−</i> bone marrow cells. The number of GFP-positive cells in <i>Inca1^−/−</i> bone marrow decreased significantly from weeks 4 and 8 until week 40 (*p = 0.015 and **p = 0.04, respectively). This analysis was restricted to mice without overt leukemia. <i>Inca1^+/+</i>: n = 14 at 4 and 8 weeks, n = 9 for 32 and 40 weeks; <i>Inca1^−/−</i>: n = 10 at 4 weeks, n = 9 at 8 and 32 weeks, n = 7 at 40 weeks. <b>C.</b> Colony assays with two subsequent replatings using AML1-ETO9a-positive <i>Inca1^+/+</i> or <i>Inca1^−/−</i> bone marrow cells, respectively, that were FACS-sorted from non-leukemic transplanted mice (n = 3 from each genotype). <i>Inca1^−/−</i> bone marrow cells transduced with AML1-ETO9a formed less colonies and replated worse than transduced <i>Inca1^+/+</i> cells (1^st plating: p = 0.01; 2^nd plating: p = 0.03; 3^rd plating: n.s.). <b>D and E.</b> For a cloning efficiency assay, 1 to 300 GFP-positive <i>Inca1^+/+</i>; AML1-ETO9a or <i>Inca1^−/−</i>; AML1-ETO9a bone marrow cells from non-leukemic transplanted mice were FACS-sorted two (<b>D</b>) or six months (<b>E</b>) and lin⁻GFP⁺ cells were seeded in semi-solid medium in a 48-well plate (n = 14 for each concentration). Two months after transplantation, <i>Inca1^+/+</i> cells had a clone forming frequency of 1/31, while the frequency was much lower in <i>Inca1^−/−</i> cells (1/164; p = 0.0001) (<b>D</b>). After six months, cloning efficiency of <i>Inca1^−/−</i> cells decreased to 1/483 (<b>E</b>).</p

Inca1 is required for AML1-ETO9a-driven leukemia initiation and maintenance <i>in vivo</i>.

<p><b>A.</b> Survival curve of recipient mice which were transplanted in two independent experiments with bone marrow cells of <i>Inca1^+/+</i> and <i>Inca1^−/−</i> mice that were retrovirally transduced with AML1-ETO9a. Left-hand side: Transplantation with 100,000 AML1-ETO9a⁺ cells (both genotypes: n = 15); right-hand side: Transplantation with 250,000 AML1-ETO9a⁺ cells (<i>Inca1^+/+</i>: n = 14, <i>Inca1^−/−</i>: n = 10). Only one <i>Inca1^−/−</i> bone marrow transplanted with 250,000 AML1-ETO9a⁺ cells led to a lethal leukemic phenotype, while about half of the <i>Inca1^+/+</i> AML1-ETO9a transplanted mice died within 300 days. <b>B.</b> Bone marrow smears (upper panel) and spleen sections (lower panel) of <i>Inca1^+/+</i> and <i>Inca1^−/−</i> AML1-ETO9a transplanted mice that died of a leukemic phenotype. Arrows in the upper panels hint at leukemic blasts, indicating acute myeloid leukemia. HE stained sections revealed morphological disruption of the splenic structure and accumulation of myeloid cells in both genotypes (lower panels). <b>C.</b> Leukemic phenotypes of recipients that received AML1-ETO9a-transduced <i>Inca1^+/+</i> and <i>Inca1^−/−</i> bone marrow cells are characterized by an infiltration of bone marrow and spleen with GFP⁺c-kit⁺ leukemic blasts in both genotypes. <b>D.</b> Survival curve of secondary recipient mice which were transplanted with bone marrow cells of leukemic mice derived from the primary transplantation shown in Fig. 5A. All mice transplanted with primary leukemic <i>Inca1^+/+</i>; AML1-ETO9a bone marrow cells died within 60 days, whereas secondary recipients of <i>Inca1^−/−</i>; AML1-ETO9a cells died with an increased latency of 150 days (<i>Inca1^+/+</i>: n = 18, <i>Inca1^−/−</i>: n = 5; p = 0.001).</p

Absence of <i>Inca1</i> accelerates MLL-AF9 driven murine leukemogenesis.

<p><b>A.</b> Survival curves of recipient mice, which were transplanted with bone marrow cells of <i>Inca1^+/+</i> or <i>Inca1^−/−</i> mice that were retrovirally transduced with MLL-AF9 as depicted in Fig. 4A (n = 11 of each genotype). Cells of both genotypes led to a fatal leukemic disease with comparable latency. <b>B.</b> Survival curves of secondary recipient mice which were transplanted with 10⁶ GFP⁺ leukemic spleen cells of leukemic mice derived from the primary transplantation shown in Fig. 5B. The secondary recipients of <i>Inca1^−/−</i>; MLL-AF9 cells (n = 15) died after a significantly longer latency than mice transplanted with <i>Inca1^+/+</i>; MLL-AF9 primary blasts (n = 15; p<0.001). Secondary transplantation of <i>p16^−/−</i>; MLL-AF9 blasts also had elongated survival compared to wild type-MLL-AF9 cells (p<0.001), but to a significant less extent (<i>Inca1^−/−</i> vs <i>p16^−/</i>−: p<0.001). <b>C.</b> Colony assays using MLL-AF9/GFP⁺c-kit⁺<i>Inca1^+/+</i> or <i>Inca1^−/−</i> spleen blast, respectively. <i>Inca1^−/−</i>; MLL-AF9 blasts formed less colonies <i>Inca1^+/+</i> blasts (p = 0.05, t-test). <b>D.</b> For a cloning efficiency assay, <i>Inca1^+/+</i>; MLL-AF9 or <i>Inca1^−/−</i>; MLL-AF9 bone marrow cells from leukemia-transplanted mice were FACS-sorted and 1 to 300 c-kit⁺GFP⁺ cells were seeded in semi-solid medium in a 48-well plate. <i>Inca1^+/+</i> cells had a clone forming frequency of 1/13, while the frequency was much lower in <i>Inca1^−/−</i> cells (1/54; p = 0.0001). Shown here are the mean results of two independent experiments.</p

Dll4 and Notch signalling couples sprouting angiogenesis and artery formation.

Endothelial sprouting and proliferation are tightly coordinated processes mediating the formation of new blood vessels during physiological and pathological angiogenesis. Endothelial tip cells lead sprouts and are thought to suppress tip-like behaviour in adjacent stalk endothelial cells by activating Notch. Here, we show with genetic experiments in postnatal mice that the level of active Notch signalling is more important than the direct Dll4-mediated cell-cell communication between endothelial cells. We identify endothelial expression of VEGF-A and of the chemokine receptor CXCR4 as key processes controlling Notch-dependent vessel growth. Surprisingly, genetic experiments targeting endothelial tip cells in vivo reveal that they retain their function without Dll4 and are also not replaced by adjacent, Dll4-positive cells. Instead, activation of Notch directs tip-derived endothelial cells into developing arteries and thereby establishes that Dll4-Notch signalling couples sprouting angiogenesis and artery formation

Dll4 and Notch signalling couples sprouting angiogenesis and artery formation.

Endothelial sprouting and proliferation are tightly coordinated processes mediating the formation of new blood vessels during physiological and pathological angiogenesis. Endothelial tip cells lead sprouts and are thought to suppress tip-like behaviour in adjacent stalk endothelial cells by activating Notch. Here, we show with genetic experiments in postnatal mice that the level of active Notch signalling is more important than the direct Dll4-mediated cell-cell communication between endothelial cells. We identify endothelial expression of VEGF-A and of the chemokine receptor CXCR4 as key processes controlling Notch-dependent vessel growth. Surprisingly, genetic experiments targeting endothelial tip cells in vivo reveal that they retain their function without Dll4 and are also not replaced by adjacent, Dll4-positive cells. Instead, activation of Notch directs tip-derived endothelial cells into developing arteries and thereby establishes that Dll4-Notch signalling couples sprouting angiogenesis and artery formation

First epileptic seizure and epilepsies in adulthood. Abridged version ofthe S2k guideline of the German Society for Neurology in cooperationwith the German Society for Epileptology

Holtkamp M, May T, Berkenfeld R, et al. Erster epileptischer Anfall und Epilepsien im Erwachsenenalter. Kurzfassung S2k-Leitlinie der Deutschen Gesellschaft für Neurologie in Zusammenarbeit mit der Deutschen Gesellschaft für Epileptologie. Clinical Epileptology. 2024.The new S2k guideline "First epileptic seizure and epilepsies in adulthood" provides recommendations on clinically relevant issues in five major topics: management of first epileptic seizures, pharmacotherapy, epilepsy surgery, complementary and supportive treatment, and psychosocial aspects. For the topic management of first epileptic seizures, the guideline provides recommendations on identifying the two major differential diagnoses, syncope and psychogenic non-epileptic seizure. The importance of additional examinations such as EEG, MRI and cerebrospinal fluid for syndromic classification and etiological allocation is discussed. Recommendations on neuropsychological and psychiatric screening tests are also given. The topic pharmacotherapy issues recommendations on antiseizure medication in monotherapy for focal, generalized and unclassified epilepsies; patient groups with special challenges such as the aged, women of childbearing potential and people with mental retardation are emphasized. Further issues are indications for measuring serum concentrations of antiseizure medication and possible risks of switching manufacturers. In the topic epilepsy surgery, indications for presurgical assessment and the multiple therapeutic approaches, such as resection, laser ablation, and neurostimulation are presented. Recommendations on postoperative management of patients, including rehabilitation and psychosocial counselling, are given. The topic complementary and supportive therapeutic approaches comprises recommendations on the diagnostics and treatment of common psychiatric comorbidities of epilepsy, such as anxiety disorder, depression and psychosis. Another important issue is the management of psychogenic non-epileptic seizures as a neuropsychiatric differential diagnosis or comorbidity of epileptic seizures. Furthermore, recommendations on the potential role of ketogenic diet and on acupuncture, homeopathy and other complementary approaches are made. The recommendations on psychosocial aspects comprise practical issues, such as fitness to drive a car, training and occupation, medical rehabilitation, sport, transition, patients' self-help, education programs for patients and next of kin, adherence, advise on SUDEP